Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

DNA-binding factors shape the mouse methylome at distal regulatory regions

A Corrigendum to this article was published on 25 April 2012

This article has been updated


Methylation of cytosines is an essential epigenetic modification in mammalian genomes, yet the rules that govern methylation patterns remain largely elusive. To gain insights into this process, we generated base-pair-resolution mouse methylomes in stem cells and neuronal progenitors. Advanced quantitative analysis identified low-methylated regions (LMRs) with an average methylation of 30%. These represent CpG-poor distal regulatory regions as evidenced by location, DNase I hypersensitivity, presence of enhancer chromatin marks and enhancer activity in reporter assays. LMRs are occupied by DNA-binding factors and their binding is necessary and sufficient to create LMRs. A comparison of neuronal and stem-cell methylomes confirms this dependency, as cell-type-specific LMRs are occupied by cell-type-specific transcription factors. This study provides methylome references for the mouse and shows that DNA-binding factors locally influence DNA methylation, enabling the identification of active regulatory regions.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1: Features of the mouse ES cell methylome.
Figure 2: General features of LMRs.
Figure 3: DNA binding is necessary and sufficient for LMR formation.
Figure 4: REST is required for LMR formation at its binding sites.
Figure 5: Methylation dynamics during differentiation.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Data sets generated for this study are available from GEO under accession GSE30206.

Change history

  • 25 April 2012

    Nature 480, 490–495 (2011) In the original version of this Article Erik van Nimwegen (Biozentrum of the University of Basel and Swiss Institute of Bioinformatics, Klingelbergstrasse 50-70, CH 4056 Basel, Switzerland) was inadvertently omitted from the author list. In the ‘Author contribution’ section the sentence beginning “Bioinformatic and statistical analyses.


  1. Bird, A. DNA methylation patterns and epigenetic memory. Genes Dev. 16, 6–21 (2002)

    Article  CAS  Google Scholar 

  2. Reik, W. Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 447, 425–432 (2007)

    Article  ADS  CAS  Google Scholar 

  3. Lister, R. et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462, 315–322 (2009)

    Article  ADS  CAS  Google Scholar 

  4. Meissner, A. et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454, 766–770 (2008)

    Article  ADS  CAS  Google Scholar 

  5. Bibel, M., Richter, J., Lacroix, E. & Barde, Y. A. Generation of a defined and uniform population of CNS progenitors and neurons from mouse embryonic stem cells. Nature Protocols 2, 1034–1043 (2007)

    Article  CAS  Google Scholar 

  6. Lienert, F. et al. Genomic prevalence of heterochromatic H3K9me2 and transcription do not discriminate pluripotent from terminally differentiated cells. PLoS Genet. 7, e1002090 (2011)

    Article  CAS  Google Scholar 

  7. Mohn, F. et al. Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol. Cell 30, 755–766 (2008)

    Article  CAS  Google Scholar 

  8. Keane, T. M. et al. Mouse genomic variation and its effect on phenotypes and gene regulation. Nature 477, 289–294 (2011)

    Article  ADS  CAS  Google Scholar 

  9. Borgel, J. et al. Targets and dynamics of promoter DNA methylation during early mouse development. Nature Genet. 42, 1093–1100 (2010)

    Article  CAS  Google Scholar 

  10. Birney, E. et al. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447, 799–816 (2007)

    Article  ADS  CAS  Google Scholar 

  11. Gross, D. S. & Garrard, W. T. Nuclease hypersensitive sites in chromatin. Annu. Rev. Biochem. 57, 159–197 (1988)

    Article  CAS  Google Scholar 

  12. Sabo, P. J. et al. Genome-wide identification of DNaseI hypersensitive sites using active chromatin sequence libraries. Proc. Natl Acad. Sci. USA 101, 4537–4542 (2004)

    Article  ADS  CAS  Google Scholar 

  13. Heintzman, N. D. et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nature Genet. 39, 311–318 (2007)

    Article  CAS  Google Scholar 

  14. Rada-Iglesias, A. et al. A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470, 279–283 (2011)

    Article  ADS  CAS  Google Scholar 

  15. Ohlsson, R., Bartkuhn, M. & Renkawitz, R. CTCF shapes chromatin by multiple mechanisms: the impact of 20 years of CTCF research on understanding the workings of chromatin. Chromosoma 119, 351–360 (2010)

    Article  CAS  Google Scholar 

  16. Kagey, M. H. et al. Mediator and cohesin connect gene expression and chromatin architecture. Nature 467, 430–435 (2010)

    Article  ADS  CAS  Google Scholar 

  17. Pastor, W. A. et al. Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature 473, 394–397 (2011)

    Article  ADS  CAS  Google Scholar 

  18. Stroud, H., Feng, S., Morey Kinney, S., Pradhan, S. & Jacobsen, S. E. 5-Hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells. Genome Biol. 12, R54 (2011)

    Article  CAS  Google Scholar 

  19. Szulwach, K. E. et al. Integrating 5-hydroxymethylcytosine into the epigenomic landscape of human embryonic stem cells. PLoS Genet. 7, e1002154 (2011)

    Article  CAS  Google Scholar 

  20. Williams, K. et al. TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity. Nature 473, 343–348 (2011)

    Article  ADS  CAS  Google Scholar 

  21. Wu, H. et al. Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells. Nature 473, 389–393 (2011)

    Article  ADS  CAS  Google Scholar 

  22. Kim, T. H. et al. Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome. Cell 128, 1231–1245 (2007)

    Article  CAS  Google Scholar 

  23. Wendt, K. S. et al. Cohesin mediates transcriptional insulation by CCCTC-binding factor. Nature 451, 796–801 (2008)

    Article  ADS  CAS  Google Scholar 

  24. Cohen, N. M. et al. DNA methylation programming and reprogramming in primate embryonic stem cells. Genome Res. 19, 2193–2201 (2009)

    Article  CAS  Google Scholar 

  25. Lienert, F. et al. Identification of genetic elements that autonomously determine DNA methylation states. Nature Genet. 43, 1091–1097 (2011)

    Article  CAS  Google Scholar 

  26. Tsumura, A. et al. Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Genes Cells 11, 805–814 (2006)

    Article  CAS  Google Scholar 

  27. Bell, A. C. & Felsenfeld, G. Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature 405, 482–485 (2000)

    Article  ADS  CAS  Google Scholar 

  28. Hark, A. T. et al. CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 405, 486–489 (2000)

    Article  ADS  CAS  Google Scholar 

  29. Jorgensen, H. F., Chen, Z. F., Merkenschlager, M. & Fisher, A. G. Is REST required for ESC pluripotency? Nature 457, E4–E5 (2009)

    Article  ADS  CAS  Google Scholar 

  30. Splinter, E. et al. CTCF mediates long-range chromatin looping and local histone modification in the β-globin locus. Genes Dev. 20, 2349–2354 (2006)

    Article  CAS  Google Scholar 

  31. Bibel, M. et al. Differentiation of mouse embryonic stem cells into a defined neuronal lineage. Nature Neurosci. 7, 1003–1009 (2004)

    Article  CAS  Google Scholar 

  32. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006)

    Article  CAS  Google Scholar 

  33. Kinoshita, K. et al. GABPα regulates Oct-3/4 expression in mouse embryonic stem cells. Biochem. Biophys. Res. Commun. 353, 686–691 (2007)

    Article  CAS  Google Scholar 

  34. Sohn, J. et al. Identification of Sox17 as a transcription factor that regulates oligodendrocyte development. J. Neurosci. 26, 9722–9735 (2006)

    Article  CAS  Google Scholar 

  35. Chen, X. et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133, 1106–1117 (2008)

    Article  CAS  Google Scholar 

  36. Bryne, J. C. et al. JASPAR, the open access database of transcription factor-binding profiles: new content and tools in the 2008 update. Nucleic Acids Res. 36, D102–D106 (2008)

    Article  CAS  Google Scholar 

  37. Illingworth, R. S. & Bird, A. P. CpG islands – ‘a rough guide’. FEBS Lett. 583, 1713–1720 (2009)

    Article  CAS  Google Scholar 

  38. Hodges, E. et al. Directional DNA methylation changes and complex intermediate states accompany lineage specificity in the adult hematopoietic compartment. Mol. Cell 44, 17–28 (2011)

    Article  CAS  Google Scholar 

  39. Irizarry, R. A. et al. The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores. Nature Genet. 41, 178–186 (2009)

    Article  CAS  Google Scholar 

  40. Brunk, B. P., Goldhamer, D. J. & Emerson, C. P., Jr Regulated demethylation of the myoD distal enhancer during skeletal myogenesis. Dev. Biol. 177, 490–503 (1996)

    Article  CAS  Google Scholar 

  41. Mareš, J. et al. Methylation changes in promoter and enhancer regions of the WT1 gene in Wilms’ tumours. Cancer Lett. 166, 165–171 (2001)

    Article  Google Scholar 

  42. Sharrard, R. M., Royds, J. A., Rogers, S. & Shorthouse, A. J. Patterns of methylation of the c-myc gene in human colorectal cancer progression. Br. J. Cancer 65, 667–672 (1992)

    Article  CAS  Google Scholar 

  43. Tagoh, H. et al. Dynamic reorganization of chromatin structure and selective DNA demethylation prior to stable enhancer complex formation during differentiation of primary hematopoietic cells in vitro. Blood 103, 2950–2955 (2004)

    Article  CAS  Google Scholar 

  44. Thomassin, H., Flavin, M., Espinas, M. L. & Grange, T. Glucocorticoid-induced DNA demethylation and gene memory during development. EMBO J. 20, 1974–1983 (2001)

    Article  CAS  Google Scholar 

  45. Groudine, M. & Conkin, K. F. Chromatin structure and de novo methylation of sperm DNA: implications for activation of the paternal genome Science. 228, 1061–1068 (1985)

  46. Colaneri, A. et al. Expanded methyl-sensitive cut counting reveals hypomethylation as an epigenetic state that highlights functional sequences of the genome. Proc. Natl Acad. Sci. USA 108, 9715–9720 (2011)

    Article  ADS  CAS  Google Scholar 

  47. Boyle, A. P. & Furey, T. S. High-resolution mapping studies of chromatin and gene regulatory elements. Epigenomics 1, 319–329 (2009)

    Article  CAS  Google Scholar 

  48. Abbott, A. Europe to map the human epigenome. Nature 477, 518 (2011)

    Article  ADS  CAS  Google Scholar 

  49. Satterlee, J. S., Schubeler, D. & Ng, H. H. Tackling the epigenome: challenges and opportunities for collaboration. Nature Biotechnol. 28, 1039–1044 (2010)

    Article  CAS  Google Scholar 

  50. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)

    Article  Google Scholar 

Download references


The authors thank A. Fernandez, C. Kohler, V. Petitjean and F. Staedtler (Novartis) and I. Nissen and C. Beisel (ETH-BSSE) for performing next generation sequencing experiments. R. Lister and J. Ecker for technical advice on BisSeq library generation. D. Schmitz for the pcDNA6-IRES-Blasticidin vector. D. Anderson for the REST antibody. H. Jørgensen for REST cds. L. Hoerner for help in Sanger bisulphite sequencing. M. Lorincz, N. Thomä and members of the Schübeler laboratory for feedback on the manuscript. R.M. is supported by an EMBO long-term postdoctoral fellowship. V.K.T. is supported by a Marie Curie International Incoming fellowship and an EMBO long-term postdoctoral fellowship. Research in the laboratory of D.S. is supported by the Novartis Research Foundation, the European Union (NoE “EpiGeneSys” FP7-HEALTH-2010-257082), the European Research Council (ERC EpiGePlas), the SNF Sinergia program and the Swiss initiative in Systems Biology (Cell Plasticity).

Author information

Authors and Affiliations



Experiments were designed by R.M., F.L., A.S., V.K.T., E.J.O. and D.S. BisSeq, RNA-Seq and ChIP-seq experiments were conducted by R.M., A.S. and V.K.T. ChIP-seq data analysis was performed by M.B.S. and L.B. BS-PCR validation was performed by R.M., F.L. and C.W. Sequencing data processing was performed by D.G. and M.B.S. LMRs were first noticed by D.G. Bioinformatic and statistical analyses were conducted by M.B.S., L.B., R.I. and E.v.N. The manuscript was prepared by R.M., M.B.S., L.B. and D.S.

Corresponding author

Correspondence to Dirk Schübeler.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-16 with legends, Supplementary Methods and additional references. The methods in this file were replaced on 25 April 2012. (PDF 16260 kb)

Supplementary Table 1

The table displays details of sequence datasets used in this study, and additional references (for external data sets only). (DOC 56 kb)

Supplementary Table 2

The table displays methylation segments identified in ES cells. (CSV 9880 kb)

Supplementary Table 3

The table displays Methylation segments identified in NP. A short description of each column is given at the top of the table. (CSV 7235 kb)

Supplementary Table 4

The table displays Genotype structure of the ES cell line used in the study. (CSV 29 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Stadler, M., Murr, R., Burger, L. et al. DNA-binding factors shape the mouse methylome at distal regulatory regions. Nature 480, 490–495 (2011).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing